title: A Ca2+-sensing molecular switch based on alternate frame protein folding.
abstract: Existing strategies for creating biosensors mainly rely on large conformational changes to transduce a binding event to an output signal. Most molecules, however, do not exhibit large-scale structural changes upon substrate binding. Here, we present a general approach (alternate frame folding, or AFF) for engineering allosteric control into ligand binding proteins. AFF can in principle be applied to any protein to establish a binding-induced conformational change, even if none exists in the natural molecule. The AFF design duplicates a portion of the amino acid sequence, creating an additional "frame" of folding. One frame corresponds to the wild-type sequence, and folding produces the normal structure. Folding in the second frame yields a circularly permuted protein. Because the two native structures compete for a shared sequence, they fold in a mutually exclusive fashion. Binding energy is used to drive the conformational change from one fold to the other. We demonstrate the approach by converting the protein calbindin D(9k) into a molecular switch that senses Ca2+. The structures of Ca2+-free and Ca2+-bound calbindin are nearly identical. Nevertheless, the AFF mechanism engineers a robust conformational change that we detect using two covalently attached fluorescent groups. Biological fluorophores can also be employed to create a genetically encoded sensor. AFF should be broadly applicable to create sensors for a variety of small molecules.
authors.affiliation: Department of Biochemistry & Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Stratton
authors.forename: Diana M
authors.initials: DM
authors.lastname: Mitrea
authors.forename: Stewart N
authors.initials: SN
authors.lastname: Loh
mesh_terms: D000494:Allosteric Regulation; D015374:Biosensing Techniques / Q000379:methods*; D064026:Calbindins; D002118:Calcium / Q000032:analysis* / Q000494:pharmacology; D008024:Ligands; D011487:Protein Conformation / Q000187:drug effects; D015202:Protein Engineering; D017510:Protein Folding; D064030:S100 Calcium Binding Protein G / Q000737:chemistry
chemical_list: D064026:Calbindins; D008024:Ligands; D064030:S100 Calcium Binding Protein G; D002118:Calcium